Glycoproteins are integral components of the plasma membrane of animal cells. While the addition of the terminal glycose moieties to these proteins is known to occur by stepwise transfer from a sugar-nucleotide donor to the appropriate acceptor, neither all the discrete steps involved in the biosynthesis of the core structure and the transfer of this core to the protein nor the regulation of these processes are yet understood. Lipid-linked saccharides of mannose, N-acetylglucosamine, and glucose are intermediates in the biosynthesis of this glycose core region. I propose to isolate and biochemical characterize glycosylation mutants of Chinese hamster ovary cells with specific lesions in the biosynthesis of the core region of membrane glycoproteins. Following characterization of these mutants, the effect of the defect on the cellular glycoproteins and on the biology of the cell will be examined. Specifically, our approach involves the following three steps. First, we have already shown using a series of in vitro and in vivo labelling procedures that are glycosylation-deficient cell line lacks the ability to synthesize glucosyloligosaccharide-lipid. This cell line was isolated during a selection procedure using concanavalin A, a cytotoxic lectin specific for alpha-mannosyl and alpha-glucosyl residues. We plan to determine the consequences of the glycosylation defect, now thought to be the last step of oligosaccharide-lipid biosynthesis, on the glycoproteins which are produced by these cells. Second, six other putative glycosylation mutants, already isolated using both selection or screening procedures, will be characterized. Finally, additional mutants will be sought using a screen which will be developed to detect cells deficient in the enzyme(s) which transfer the glycose moieties from lipid to proteins.